A New Orchid Genus, Danxiaorchis, and PhylogeneticAnalysis of the Tribe CalypsoeaeJun-Wen Zhai1,2,3, Guo-Qiang Zhang2,8, Li-Jun Chen2, Xin-Ju Xiao2, Ke-Wei Liu2,7, Wen-Chieh Tsai4, Yu-

Yun Hsiao4, Huai-Zhen Tian5, Jia-Qiang Zhu6, Mei-Na Wang2, Fa-Guo Wang1, Fu-Wu Xing1*, Zhong-

Jian Liu2,7,8,9*

1 South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China, 2 Shenzhen Key Laboratory for Orchid Conservation and Utilization, The National

Orchid Conservation Center of China and The Orchid Conservation and Research Center of Shenzhen, Shenzhen, China, 3Graduate University of Chinese Academy of

Sciences, Beijing, China, 4 Institute of Tropical Plant Sciences and Orchid Research Center, National Cheng Kung University, Tainan City, Taiwan, 5 School of Life Science,

East China Normal University, Shanghai, China, 6 Information Center of Renhua County, Shaoguan, China, 7Center for Biotechnology and BioMedicine, Graduate School at

Shenzhen, Tsinghua University, Shenzhen, China, 8 Landscape College of Fujian Agriculture and Forestry University, Fuzhou, China, 9College of Forestry, South China

Agricultural University, Guangzhou, China

Abstract

Background: Orchids have numerous species, and their speciation rates are presumed to be exceptionally high, suggestingthat orchids are continuously and actively evolving. The wide diversity of orchids has attracted the interest of evolutionarybiologists. In this study, a new orchid was discovered on Danxia Mountain in Guangdong, China. However, the phylogeneticclarification of this new orchid requires further molecular, morphological, and phytogeographic analyses.

Methodology/Principal Findings: A new orchid possesses a labellum with a large Y-shaped callus and two sacs at the base,and cylindrical, fleshy seeds, which make it distinct from all known orchid genera. Phylogenetic methods were applied toa matrix of morphological and molecular characters based on the fragments of the nuclear internal transcribed spacer,chloroplast matK, and rbcL genes of Orchidaceae (74 genera) and Calypsoeae (13 genera). The strict consensus Bayesianinference phylogram strongly supports the division of the Calypsoeae alliance (not including Dactylostalix andEphippianthus) into seven clades with 11 genera. The sequence data of each species and the morphological charactersof each genus were combined into a single dataset. The inferred Bayesian phylogram supports the division of the 13 generaof Calypsoeae into four clades with 13 subclades (genera). Based on the results of our phylogenetic analyses, Calypsoeae,under which the new orchid is classified, represents an independent lineage in the Epidendroideae subfamily.

Conclusions: Analyses of the combined datasets using Bayesian methods revealed strong evidence that Calypsoeae isa monophyletic tribe consisting of eight well-supported clades with 13 subclades (genera), which are all in agreement withthe phytogeography of Calypsoeae. The Danxia orchid represents an independent lineage under the tribe Calypsoeae of thesubfamily Epidendroideae. This lineage should be treated as a new genus, which we have named Danxiaorchis, that isparallel to Yoania. Both genera are placed under the subtribe Yoaniinae.

Citation: Zhai J-W, Zhang G-Q, Chen L-J, Xiao X-J, Liu K-W, et al. (2013) A New Orchid Genus, Danxiaorchis, and Phylogenetic Analysis of the TribeCalypsoeae. PLoS ONE 8(4): e60371. doi:10.1371/journal.pone.0060371

Editor: Maria Anisimova, Swiss Federal Institute of Technology (ETH Zurich), Switzerland

Received November 25, 2012; Accepted February 12, 2013; Published April 4, 2013

Copyright: � 2013 Zhai et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The work was supported by fellowships of the Forestry Construction of State Forestry Administration of China (No. 2010–240) and the ForestryConstruction of Guangdong Province, China (No. 2010–317). The funders had no role in the study design, data collection and analysis, decision to publish, orpreparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: xinfw@scib.ac.cn (FWX); liuzj@sinicaorchid.org (Z-JL)

Introduction

Orchidaceae is one of the largest families of angiosperms [1]. It

has been said ‘‘The speciation rate in orchids is frequent because

of their diverse flower and vegetable morphologies’’ [2]. Consider-

able attention has been given to their extraordinary pollination,

multiple adaptive strategies to various habitats, and numerous

dust-like seeds that lack endosperms [3,4]. Previously, Orchida-

ceae has been divided into five subfamilies based on their

morphological characters as follows: Apostasioideae, Cypripedioi-

deae, Spiranthoideae, Orchidoideae, and Epidendroideae [5].

However, a phylogenetic analysis based on the internal transcribed

spacer (ITS), trnL-F, and matK sequences revealed that Spir-

anthoideae is a member of Orchidoideae and that Vanilla and its

allies should be separated from Epidendroideae to form a new

subfamily, Vanilloideae [6–8]. Epidendroideae is a highly evolved

and diverse subfamily, in which a few species are mycotrophic and

lack green leaves. Many holomycotrophic orchids are found in

China, comprising approximately 20 genera. None of these

orchids has a bisaccate labellum.

Vanilla, Apostasia, Cyrtosia, Palmorchis, Selenipedium, and several

Neuwiedia species have wingless seeds with hard seed coats. Several

members of Vanilleae, such as Epistephium and Galeola, have a hard

seed coat over the embryo and a developed wing around the seed.

Several Neuwiedia species have small seeds with sac-like appendages

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at either end. Most other orchids have a loose, rather papery seed

coat around the embryo, which has a length that ranges from

0.15 mm to 6 mm. However, all of these orchid seeds are dry and

lack an endosperm.

In this report, we documented a new orchid found on Danxia

Mountain in Guangdong, China. The flower and seed structures

of this new orchid are different from those of other known taxa in

Orchidaceae. However, the phylogenetic clarification of this new

orchid requires further molecular, morphological, and phytogeo-

graphic analyses.

Results

Morphological AnalysisThe new orchid entity is restricted to the Danxia Mountain in

northern Guangdong, China (Fig. S1). The Danxia region, known

as the Danxia Landform, is famous for its topographic features. A

detailed comparison between the newly discovered orchid and

other members of Orchidaceae was conducted. The new plant is

characterized by a labellum with two sacs at the base, an elongated

column that has a terminal concave stigma and lacks conspicuous

staminodes and rostellum, four sectile pollinia attached by two

caudicles to a common large viscidium, and cylindrical, fleshy

seeds. These features distinguish the new orchid from all other

known orchids (Figs. 1, 2, and S2).

Analyses of Phylogenetic PlacementDanxiaorchis singchiana is morphologically related to the tribe

Calypsoeae and, to a lesser degree, to the tribe Gastrodieae. Both

of these tribes belong to the subfamily Epidendroideae. A detailed

morphological character matrix (59 characters of 74 taxa) was

integrated with a molecular matrix (3586 nucleotide sequences of

the ITS, matK, and rbcL genes of 74 genera) to classify the plant

into an appropriate phylogenetic position (Figs. 3, S3, S4, and S5).

Bayesian inference (BI) phylogram showed the monophyly of

the new orchid plant. Five clades were distinguished in

Orchidaceae, with a posterior probability (PP) of over 99%

(Fig. 3). Based on evolutionary sequences, the five clades

correspond to the subfamilies Apostasioideae, Cypripedioideae,

Vanilloideae, Orchidoideae, and Epidendroideae. Calypsoeae

formed an independent lineage (PP=52%) in the subfamily

Epidendroideae, under which Danxiaorchis is included together

with other genera of Calypsoeae. In the family-level BI phylogram

of the combined ITS, matK, and rbcL gene sequences, the

Calypsoeae clade is divided into two subclades (PP= 95%, Fig.

S4). The first subclade includes Calypso, Tipularia, and Changnienia,

whereas the second subclade comprises eight genera, including

Danxiaorchis, which is most closely related to Yoania, as confirmed

by maximum parsimony (MP) analysis (Fig. S5).

Phylogeny of CalypsoeaeNuclear ITS sequence data analysis. The phylogenetic

trees generated based on the ITS sequence data analysis clearly

revealed the independence of the eight genera of this tribe. The BI

phylogram with most of the clades received a strong support

(PP.90%). Danxiaorchis, which forms a single clade with a PP of

99%, has been recognized as a natural genus within this tribe (Fig.

S6). However, a relatively weak bootstrap and unstable topology is

found in MP phylogram (Fig. S7).

Chloroplast sequence data analysis. Similarly, the 11

genera can be easily distinguished from the phylograms based on

chloroplast sequence data analysis. The phylogenetic topologies

generated by BI are approximately congruent with the ones by MP

analysis (Figs. S8 and S9). The basal clade is independently

composed of Calypso, Tipularia, and Changnienia. The next clade is

Govenia, followed by a complex clade, which includes Aplectrum,

Cremastra, Danxiaorchis, Yoania, Wullschlaegelia, and Oreorchis. Cor-

allorhiza occupies the terminal positions in both MP and BI

phylograms, although it is not well-supported intragenetically in

the MP phylogram.

Combined analysis. In this study, ITS, matK, and rbcL were

combined into a single dataset. The strict consensus BI phylogram

(Fig. S10) strongly supports the division of the Calypsoeae alliance

(except Dactylostalix and Ephippianthus) into seven clades with eight

subclades (PP= 100%, except for one with 77%). The first clade,

which consists of the Changnienia, Tipularia, and Calypso subclades, is

strongly supported as a sister to the outgroup clade, which consists

of Sobralia and Nervilia (PP= 100%). The second clade, which has

a single genus, Govenia, is strongly supported as a sister to the first

clade (PP= 100%). The third clade is the Aplectrum genus, and the

fourth is Cremastra (PP= 100%). The fifth clade contains the new

genus Danxiaorchis, and its ally, Yoania (PP= 100%). The last two

clades have weak support (PP= 77%). The sixth clade is comprised

of Wullschlaegelia and Oreorchis. The seventh clade contains a single

genus, Corallorhiza, which consists of 13 species that are further

divided into two subclades. The results are in agreement with the

results of the complex clades in the MP phylogram (Fig. S11).

The sequence data of each species and the morphological

characters of each genus were combined into a single dataset

(Dactylostalix and Ephippianthus having morphological characters

only). The strict consensus BI phylogram supports the division of

the 13 genera of Calypsoeae into four clades with 13 subclades,

which is in agreement with the results of the combined sequence

data analysis. These results show that the genera Dactylostalix and

Ephippianthus belong to a single clade near the Govenia and Calypso

clades (Figs. 4 and 5).

Discussion

Morphological AnalysisThis study is the first to report an orchid with a bisaccate

labellum and cylindrical, fleshy seeds. This orchid is difficult to

classify in any known subtribe or tribe within Orchidaceae.

Although the new orchid has a particular similarity to Gastrodia and

its allies in terms of pollinium structure, the pollinarium of Gastrodia

lacks distinct caudicles and viscidium like the new orchid does.

This new orchid sharply differs from Satyrium and Corybas except

for the two elongate or saccate spurs at the base of their labellum

[9]. Danxiaorchis has cylindrical (1.5 mm60.5 mm) and fleshy

seeds, which is similar to the seeds of underground orchid species

of Rhizanthella [10]. However they’re distinct from Danxiaorchis by

its underground habitat, absence of roots, fleshy overlapping

bracts, small flowers and minute seeds. Thus, this genus is

distinguishable from all other genera of orchids.

Family-level AnalysisThe results of our analyses are in agreement with those obtained

by previous researchers [5,9,11,12] and support the relationships

among the subfamilies Apostasioideae, Vanilloideae, Cypripedioi-

deae, Orchidoideae, and Epidendroideae. In addition, a more

precise phylogenetic tree was obtained in the present study at

higher categories. This finding may be attributed in part to the

utilization of a more diversified nuclear genetic marker, ITS, the

application of multiple genetic markers, and the integration of

morphological and molecular characters.

A New Orchid Genus of Tribe Calypsoeae

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Figure 1. Danxiaorchis singchiana flowers and seeds. (A) Flowers with a Y-shaped appendage (arrow) on the labellum. Bar = 1 cm. (B) Labellumwith two sacs (arrows) at the base. Bar = 4 mm. (C) Column and labellum, side view. Bar = 4 mm. (D). Appendage of the labellum, side view.Bar = 2 mm. (E) Pollinarium, front view, showing pollinia (red arrows), caudicles (white arrows), and viscidium (yellow arrow). Bar = 1 mm. (F) MatureSeeds, showing abortive seed (yellow arrow). Bar = 5 mm.doi:10.1371/journal.pone.0060371.g001

A New Orchid Genus of Tribe Calypsoeae

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Calypsoeae-level AnalysisDressler [5] and Pridgeon et al. [ed.] [13] investigated the tribe

Calypsoeae but did not propose any subtribe. Dressler defined

Calypsoeae as comprising nine genera, namely, Aplectrum, Calypso,

Corallorhiza, Cremastra, Dactylostalix, Ephippianthus, Oreorchis, Tipularia,

and Yoania. Pridgeon et al. [ed.] [13] added Wullschlaegelia and

Govenia to this tribe, although Dressler had placed the former in

Gastrodiinae and the latter in Cymbidieae [5], both at subtribal

rank. Meanwhile, Chen et al. [14] treated these genera as

members of the tribe Epidendreae, with the following subtribes

in China: subtribe Yoaniinae with the genus Yoania; subtribe

Calypsoinae with the genera Oreorchis, Cremastra, Tipularia, Calypso,

and Changnienia; and subtribe Corallorhizinae with the genus

Corallorhiza. However, all of these classification systems were based

only on morphological evidence.

Govenia and Corallorhiza are both monophyletic genera

(PP= 100%), with the former genus having a relatively anomalous

distribution in the tribe Calypsoeae [13]. Thus, their subtribal

rank, the subtribes Goveniinae [5] and Corallorhizinae [14], is

maintained. The phylogenetic placement and the infrageneric

relationships of Corallorhizinae are very similar to those studied by

Freudenstein et al. [15] Corallorhiza should be divided into two

subclades. The first subclade contains C. striata Lindl., C. bentleyi

Freudenst., C. involuta Greenm., and C. vreelandii Rydb. The species

of this subclade possess three-veined perianth segments and

a thickened labellum that are fused at the base. The second

subclade contains the rest of the species in this genus. They possess

a thin-textured labellum, and some species, such as C. trifida

Chatel. and C. odontorhiza (Willd.) Nutt. are autogamous [15–18].

Aplectrum and Wullschlaegelia are composed of a few species that

are distributed from North America to tropical South America.

For Wullschlaegelia, a monotypic subtribe Wullschlaegeliinae was

established in 1990 [5]. Cremastra and Oreorchis are only found in

Asia [19,20]. Yoania is most closely related to Danxiaorchis and they

share the same habitat. However, Danxiaorchis can be distinguished

from Yoania by its rooted rhizome, bisaccate labellum, Y-shaped

appendages, and caudicles [21].

The Danxia Mountain located in northern Guangdong, where

Danxiaorchis grows, was formed approximately 6 million years ago

[22]. The unique geological conditions and the relative environ-

mental isolation of the Danxia Mountain might have favored the

speciation of new taxa, such as Oberonioides microtatantha (Schltr.)

Szlach. [14], Firmiana danxiaensis H. H. Hsue et H. S. Kiu [23], and

Lyonia danxiaensis Miau et W. Q. Liu [24].

Two distantly related genera, Dactylostalix and Ephippianthus [13],

are distributed along the Sakhalin Peninsula in northern Japan

and the Kuriles. Unfortunately, materials from these two genera

could not be obtained. However, in our study, these two genera

formed a sister clade with the Calypso and Govenia clades based on

their morphological characters.

ConclusionThe Danxia orchid has several distinct features. Based on results

obtained by applying phylogenetic methods to a matrix of

morphological and molecular characters, the Danxia orchid can

be treated as a new genus of Calypsoeae (subfamily Epidendroi-

deae). Analysis of the combined datasets using maximum likeli-

hood methods revealed strong evidence that Calypsoeae is

a monophyletic tribe consisting of eight well-supported clades

with 13 subclades, which are all in agreement with the

phytogeography of Calypsoeae.

The Danxia orchid represents an independent lineage under the

tribe Calypsoeae of the subfamily Epidendroideae. This lineage

should be treated as a new genus parallel to Yoania under the

subtribe Yoaniinae. The new classification should be as follows:

Subfamily: Epidendroideae

Tribe: Calypsoeae

Subtribe: Yoaniinae Szlach.

Danxiaorchis singchiana J. W. Zhai, F. W. Xing, and Z. J.

Liu gen. et sp. nov. (Figs. 1, 2, and S2) [Danxiaorchis,

urn:lsid:ipni.org:names: 77124908-1; D. singchiana, urn:lsid:ip-

ni.org:names: 77124909-1].

N Etymology: The generic name alludes to Danxia, the name

of the locality where it was found. The Danxia Mountain is

famous for its topographic feature, the Danxia Landform. The

Greek name for orchid, orchis, is then incorporated. Thus,

Figure 2. Danxiaorchis singchiana, J. W. Zhai, F. W. Xing et Z. J.Liu: (1) flowering plant; (2) flower, front view; (3) column andlabellum, longitudinal section; (4) appendage of the labellum,side view; (5) seed; (6) ovary, cross section; (7) dorsal sepal,petal, and lateral sepal; (8) labellum, flattened; (9) pollinarium;(10) column, front view; (11) capsule.doi:10.1371/journal.pone.0060371.g002

A New Orchid Genus of Tribe Calypsoeae

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Figure 3. Bayesian consensus phylograms for the combined ITS, matK, and rbcL datasets and 59 morphological character matrix,including 72 genera of Orchidaceae. The Bayesian PP (6100) is provided above the branches.doi:10.1371/journal.pone.0060371.g003

A New Orchid Genus of Tribe Calypsoeae

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A New Orchid Genus of Tribe Calypsoeae

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Danxiaorchis refers to an orchid growing on the Danxia

Mountain. The specific epithet singchiana is dedicated to the

Chinese professor Sing-Chi Chen, a famous, internationally

renowned orchidologist.

N Type: Guangdong, Renhua, Danxiashan, in a forest, alt.

125 m, 2011.5.31. J. W. Zhai, 5481 (holotype, NOCC; isotype:

IBSC).

N Diagnosis: The new remarkable genus is distinct from all known

orchid genera by it possesses a labellum with a large Y-shaped callus and

two sacs at the base, and cylindrical, fleshy seeds.

N Description: Holomycotrophic plants 21 to 40 cm tall;

rhizome tuberous, fleshy, cylindrical, 5 to 6 cm long, 0.6 to

1.8 cm thick, shortly branched, rooting. Scape erect, terete,

pale red-brown, slightly tinged with green-yellow, 3- to 4-

sheathed; sheaths, cylindrical, clasping stem, membranous, 2.3

to 3.5 cm long; raceme 5 to 8.5 cm long, 2- to 13-flowered;

floral bracts oblong-lanceolate, 1.5 to 2.3 cm long; pedicel and

ovary 2.2 to 4.6 cm long, glabrous; sepals and petals pale

yellow; lip yellow, with pale purple-red stripes on side-lobes

and purple-red spots on mid-lobe; dorsal sepals narrowly

elliptic, 1.8 to 2.6 cm 6 6 to 9 mm, acute; lateral sepals

obovate-elliptic, 2 to 2.3 cm67 to 9 mm, acute; petals

narrowly elliptic, 2 to 2.2 cm66.5 to 7.5 mm, acute; labellum

3-lobed; side-lobes erect, slightly clasping the column,

subsquare, up to 5 mm long and 5.5 mm wide; mid-lobe

oblong, 7 to 8 mm65 to 8 mm, apex rounded-obtuse;

labellum with two sacs at the base and a Y-shaped fleshy

appendage centrally; appendage extending from the base of

disc to the base of mid-lobe, 1.3 to 1.5 mm tall; column semi-

terete, 5 to 7 mm long, footless; stigma concave, terminal;

anther cap ellipsoid; pollinia four, in two pairs, subobovoid-

globose, granular-farinaceous, composed of friable massulae,

each pair containing two pollinia unequal in size with a thick

caudicle attached to a common subsquare viscidium. Capsule

fusiform, 3 to 4.2 cm long, 0.8 to 1.2 cm thick. Seeds

cylindrical, 1.560.5 mm, fleshy. Fl. April–May. Fr. May–June.

Materials and Methods

MaterialsThe locations of the field studies are neither private lands nor

protected areas, but are controlled by the State Forestry

Administration of China, to which our institution is affiliated.

The State Forestry Administration authorized us to conduct

scientific observations or tests in the regions it controls.A valid

permit was also obtained for testing the genes of Danxiaorchis.

A total of 74 genera were analyzed in the family-level study.

Two genera, Hypoxis and Curculigo, were selected as outgroups.

Three genetic markers (ITS, matK, and rbcL) of Danxiaorchis,

Corallorhiza, Cremastra, Oreorchis, and Yoania were analyzed. The

gene sequences of the other 61 genera were accessed from

GenBank (Table S1). Danxiaorchis singchiana was collected from the

Danxia Mountain in northern Guangdong, China (25uN, 113uE).A total of 34 species (or subspecies or varieties) and 35

individuals of 13 genera were included in the tribe-level analysis,

wherein Sobralia and Nervilia were selected as outgroups. The ITS,

matK, and rbcL gene sequences of Danxiaorchis singchiana, Corallorhiza

trifida, Changnienia malipoensis, Cremastra appendiculata, Yoania japonica,

Oreorchis indica, and O. nana were applied in the same way as that in

the family-level study. The other sequences were accessed from

GenBank (Table S2).

Corallorhiza trifida and Oreorchis nana were collected from Huang-

long in Sichuan Province. Cremastra appendiculata was cultivated in

a nursery in Shenzhen, whereas Yoania japonica was obtained from

the herbarium of The Orchid Conservation and Research Center

of Shenzhen (NOCC, Z. J. Liu 6241).

Danxiaorchis singchiana was collected between April 2012 and

May 2012 from its habitat in northern Guangdong, China. Several

individual plants with young fruits were cultivated in our nursery

in Shenzhen for mature fruits and seeds. Fresh flowers, especially

the pollinaria, were examined using a stereoscope (Guiguang

XTL-500, China). Colour photographs, black-white drawings, and

descriptions were catelogued at the time. Molecular experiments

were performed at the Shenzhen Key Laboratory for Orchid

Conservation and Utilization of The Orchid Conservation and

Research Center of Shenzhen.

All material for morphological and molecular examinations was

kept in FAA (55% alcohol: glacial acetic acid: formalin at a ratio of

95:5:5) and allochroic silica gel.

MethodsAmplification and sequencing. Total DNA was extracted

from fresh material, silica gel-dried plant tissue, or herbarium

specimens using a modified hexadecyl trimethyl ammonium

bromide method [25].

The amplification reaction included total DNA, primers,

Mighty Amp buffer version 2.0, and Mighty Amp DNA poly-

merase (Takara Bio). The polymerase chain reaction (PCR) profile

consisted of an initial 2 min pre-melt stage at 98uC; 35 cycles of

20 s at 98uC (denaturation), 20 s at 45uC to 55uC (annealing

temperature was determined by the requirements of the primer),

and 50 s to 90 s at 68uC (extension time was determined by the

length of the target DNA region); and a final extension of 6 min to

8 min at 68uC.Amplification of the ITS, matK, and rbcL regions was separately

performed using the primer pairs ITS A and ITS B, matK-19F and

trnK-2R, and rbcL [26–28]. Other matK and rbcL primer sets were

also amplified (Table S3).

The PCR products were run on 1.5% agarose gels to check the

amplified DNA quality. Gels with target products were excised,

purified using DNA gel extraction kits (OMEGA BIO-TEK,

USA), and sequenced by Invitrogen (Shanghai).

Sequence editing and assembling. The forward and re-

verse sequences as well as electropherograms were edited and

assembled using DNASTAR (http://www.dnastar.com/). The

DNA sequences were aligned using MEGA5.05 using Muscle

method [29] and then manually adjustments were made for

inserting gaps to improve the alignments [30]. The aligned

sequences are available from the corresponding authors upon

request.

Morphological analyses. A matrix, which consists of 59

morphological characters of 74 taxa in the family-level analysis

(Morphological Character Codes S1 and Table S4) and 69

morphological characters of 35 taxa in the tribe-level analysis

(Table S5), was constructed to explore the phylogenetic positions

of the Danxiaorchis alliance by morphological classification.

Data analyses. Maximum Parsimony (MP) analyses were

performed usingPAUP* version 4.0b10 [31]. All characters were

equally weighed and unordered. The test settings included 1,000

Figure 4. Bayesian consensus phylogram for the combined ITS, matK, and rbcL datasets and 69 morphological character matrix,including 35 taxa of Calypsoeae. Bayesian PP (6100) is given above the branches.doi:10.1371/journal.pone.0060371.g004

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replications of random addition sequence and heuristic search with

tree bisection and reconnection branch swapping. Tree length,

consistency indices (CI), and retention indices (RI) are shown in

Table S6. BI analysis was performed using MrBayes3.1.2 [32].

The best-fit model for each dataset was selected using Modeltest

3.7. The model for the combined ITS, matK, and rbcL datasets was

also based on the best-fit model for each individual dataset (Tables

S7 and S8). The following settings were applied: sampling

frequency = 100; temp= 0.1; burn-in = 10,000; and the number

of Markov Chain Monte Carlo generations = 4,000,000. The first

10,000 trees were discarded as burn-in. A majority-rule consensus

phylogram was constructed based on the phylograms sampled

after the 1,000,000th generation.

Nomenclature ActsThe electronic version of this article in Portable Document

Format (PDF) in a work with an ISSN or ISBN will represent

a published work according to the International Code of

Nomenclature for algae, fungi, and plants, and hence the new

names contained in the electronic publication of a PLOS ONE

article are effectively published under that Code from the

electronic edition alone, so there is no longer any need to provide

printed copies.

In addition, new names contained in this work have been

submitted to IPNI, from where they will be made available to the

Global Names Index. The IPNI LSIDs can be resolved and the

associated information viewed through any standard web browser

by appending the LSID contained in this publication to the prefix

http://ipni.org/. The online version of this work is archived and

available from the following digital repositories: PubMed Central,

LOCKSS.

Supporting Information

Figure S1 Danxiaorchis location. Map showing the Dan-

xiaorchis locality (star) in the Danxia Landform in northern

Guangdong Province, China. The inset map shows the location

of Guangdong Province in southern China.

(TIF)

Figure S2 Danxiaorchis singchiana. (A) Flowering plants in

their habitat. Bar = 4 cm; (B) Inflorescence. Bar = 1.5 cm; (C)

Fruiting plant. Bar = 2 cm; (D) Tuberous rhizome. Bar = 6 mm.

(TIF)

Figure S3 Strict consensus phylogram of most parsi-monious phylograms based on the combined ITS, matK,and rbcL datasets and a matrix composed of 59morphological characters of 72 Orchidaceae genera.Bootstrap values of the MP analysis are indicated above the

branches. Tree length = 18095 steps; CI = 0.2354; RI = 0.5600.

(TIF)

Figure S4 Bayesian consensus phylogram for the com-bined ITS, matK, and rbcL datasets, including 71 generaof Orchidaceae. Bayesian PP (6100) is indicated above the

branches.

(TIF)

Figure S5 Strict consensus phylogram of most parsi-monious phylograms based on the combined ITS, matK,

and rbcL datasets, including 71 genera of Orchidaceae.Bootstrap values for the MP analysis are indicated above the

branches. Tree length = 10188 steps; CI = 0.3248; RI= 0.5857.

(TIF)

Figure S6 Bayesian consensus phylogram for the com-bined ITS datasets, including 26 taxa of Calypsoeae.Bayesian PP (6100) is indicated above the branches.

(TIF)

Figure S7 Strict consensus phylogram of most parsi-monious phylograms based on ITS datasets, including26 taxa of Calypsoeae. The bootstrap values of the MP analysis

are indicated above the branches. Tree length = 444 steps;

CI = 0.8153; RI = 0.6641.

(TIF)

Figure S8 Bayesian consensus phylogram for the com-bined matK and rbcL datasets, including 32 taxa ofCalypsoeae. Bayesian PP (6100) is indicated above the

branches.

(TIF)

Figure S9 Strict consensus phylogram of most parsi-monious phylograms based on the combined matK andrbcL datasets, including 32 taxa of Calypsoeae. The

bootstrap values of the MP analysis are indicated above the

branches. Tree length = 931 steps; CI = 0.8217; RI= 0.8903.

(TIF)

Figure S10 Bayesian consensus phylogram for thecombined ITS, matK, and rbcL datasets, including 33taxa of Calypsoeae. Bayesian PP (6100) is indicated above the

branches.

(TIF)

Figure S11 Strict consensus phylogram of most parsi-monious phylograms based on the ITS, matK, and rbcLdatasets, including 33 taxa of Calypsoeae. The bootstrap

values of the MP analysis are indicated above the branches. Tree

length = 1505 steps; CI = 0.7980; RI= 0.8550.

(TIF)

Table S1 Samples used in Orchidaceae gene sequencingand their information.

(DOC)

Table S2 Samples used in Calypsoeae gene sequencingand their information.

(DOC)

Table S3 Primers used in this study.

(DOC)

Table S4 Morphological data matrix for the phyloge-netic analysis.

(DOC)

Table S5 Morphological data matrix for the tribe-levelphylogenetic analysis.

(DOC)

Table S6 Statistics from the analyses of various data-sets.

(DOC)

Figure 5. Strict consensus phylogram of most parsimonious phylograms based on the ITS, matK, and rbcL datasets and 69morphological character matrix, including 35 taxa of Calypsoeae. The bootstrap values of the MP analysis are given above the branches.Tree length= 3023 steps; CI = 0.7291; RI = 0.8167.doi:10.1371/journal.pone.0060371.g005

A New Orchid Genus of Tribe Calypsoeae

PLOS ONE | www.plosone.org 9 April 2013 | Volume 8 | Issue 4 | e60371

Table S7 Best-fit model and parameter for eachOrchidaceae dataset.(DOC)

Table S8 Best-fit model and parameter for eachCalypsoeae dataset.(DOC)

Morphological Character Codes S1.

(DOC)

Acknowledgments

We would like to thank Xu-Hui Chen, Wei-Rong Liu, and Wen-Hui Rao

for their help in the field work; Xin-Lan Xu, Lin Fu, and Ru-Fang Deng

for their help with Laser Scanning Confocal Microscopy; and Yu-Yun

Zheng for helping in the manuscript preparation.

Author Contributions

Conducted taxonomic treatment: JWZ FWX ZJL LJC GQZ. Conceived

and designed the experiments: ZJL JWZ FWX. Performed the experi-

ments: JWZ LJC GQZ ZJL HZT JQZ FGW. Analyzed the data: JWZ

GQZ YYH KWL ZJL FWX WCT LJC. Contributed reagents/materials/

analysis tools: JWZ ZJL JQZ GQZ LJC XJX MNW. Wrote the paper:

JWZ ZJL.

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A New Orchid Genus of Tribe Calypsoeae

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